Influenza is a major cause of disease in humans and a source of significant morbidity and mortality worldwide with large segments of the human population affected every year. Influenza viruses can be subtyped into A, B and C. The majority of viruses that circulate in the human population are influenza A and B.
Annual vaccination is the primary strategy for preventing infections. The A strain of influenza can be further subtyped, based on the antigenic differences of the two viral surface transmembrane proteins, Haemagglutinin (HA) and Neuraminidase (NA). To date 16 HA (HA1-16) and 9 NA (NA1-9) glycoprotein subtypes of influenza A viruses have been identified. At present, two subtypes of influenza A circulate in humans (H1N1 and H3N2) [1].
On occasion, an influenza pandemic can occur when a new influenza virus emerges for which people have little or no immunity. In the past century, three influenza A strain pandemic outbreaks have caused significant human influenza-related fatalities (1918, H1N1; 1957, H2N2; 1968, H3N2) [2]. In Hong Kong in 1997, a highly pathogenic H5N1 avian influenza virus was transmitted directly from chickens to humans, causing six deaths from 18 confirmed infections [3;4]. Since this time, concern regarding an influenza pandemic has been heightened by sporadic outbreaks of pathogenic H5N1 viruses. These outbreaks have resulted in 258 cases with 153 deaths across six countries, with outbreaks from Asia through to Europe (Cambodia, China, Indonesia, Thailand, Turkey, and Vietnam) [5].
Since 1997, viruses of several other subtypes, including H2N2, H9N2, H7N7, H7N3 and H10N7, have also been implicated in human infections and consequently these subtypes also represent a significant pandemic threat. Because it is not possible to predict which subtype of influenza virus will cause the next pandemic, an ideal vaccine would protect the host from severe disease or death by eliciting an immune response that protects the host against a broad range of influenza viruses, from the same or different subtypes. However, for the reasons outlined below, the available vaccines, which rely on the induction of a neutralising antibody response (primarily against HA and NA), are highly influenza strain-specific.
The HA and NA glycoproteins of influenza viruses undergo antigenic variation as a means to escape the host immune response [6]. The presence of virus neutralising antibodies specific for the HA glycoprotein at systemic or mucosal sites protects against infection with influenza. However, as a consequence of antigenic variability the antibody response to HA is highly strain-specific, and does not recognise the HA from influenza viruses of different subtypes, or even highly divergent strains within the same subtype [7]. Cell-mediated immunity on the other hand, in particular CD8+cytotoxic T cells (CD8+CTL) is primarily responsible for clearing virus-infected cells, and thus limits the severity of, and promotes recovery from infection [8]. In contrast to HA, the internal protein targets of the cell-mediated immune response—the key ones being PB2, PA, Nucleoprotein (NP) and Matrix protein (M)—are not prone to antigenic drift and as a consequence are highly conserved. For example, the NP and M proteins of the H5N1 strains A/Indonesia/5/05 and A/Vietnam/1194/04 share approximately 94% amino acid identity with A/Puerto Rico/8/34 (as shown in FIG. 1). A/Puerto/Rico/8/34 is an H1N1 virus isolated in 1934, and the source of the structural proteins for the engineered vaccine strains, traditionally prepared by re-assortment and more recently by reverse genetics. Furthermore, there is a high degree of conservation of CTL epitopes between these different influenza subtypes. Extension of this analysis to include other virus subtypes, including H7N7 and H9N2, which also pose a potential pandemic threat, demonstrates a high degree of conservation of CTL epitopes across all A-strain viruses. Therefore, unlike the HA antibody response which is highly strain-specific, CTL responses have the potential to be broadly effective, irrespective of the influenza A-strain subtype [9]. Therefore, the ability to induce a strong CTL response is a highly desirable feature for a pandemic influenza vaccine.
The induction of CD8+ CTL, particularly in humans, has to date proven to be a significant hurdle for vaccine development. Delivery systems such as DNA and viral vectors have offered some hope, but have potential safety concerns, and in the case of DNA, generally elicit poor cellular responses, in particular CD8+ CTL responses. Additionally, viral vectors have the problem of inducing neutralising antibodies to the vector, which limits repeated use. Prime-boost combinations of DNA and live viral vector delivery are currently being evaluated, and although results have been promising in animal models, they are yet to be proven in humans. ISCOM™ vaccines have been shown in numerous animal models, to be potent inducers of both T-helper (CD4+) and CTL (CD8+) T cell responses to a wide variety of antigens, including naturally occurring immunogens and recombinant proteins [10]. An H1N1 influenza ISCOM™ vaccine has been shown to confer cross protection in mice against lethal challenge with heterologous viruses, including H2N2, H3N2, H5N1 and H9N2 viruses [11]. Furthermore, protection was shown to be dependent on both CD8+ T cells and antibody [11].
It is generally accepted that the ability of ISCOM™ vaccines to induce strong CD8+ CTL responses is largely due to the fact that the antigen is incorporated into the ISCOM™ particle [12], which results in efficient cellular uptake and subsequent access of the antigen to the MHC Class I processing machinery [13]. However, the manufacture of ISCOM™ vaccines is complex, difficult to scale up, and there are significant problems associated with manufacturing control and consistency. Therefore ISCOM™ vaccines, despite demonstrating protection against a range of pathogens in a wide variety of animal species, have limited product potential, particularly for high volume products such as pandemic influenza vaccine which would demand the production of hundreds of millions of doses in a short time frame.
In work leading to the present invention, the inventors have developed a vaccine formulation in which preformed ISCOMATRIX™ adjuvant which is “immunogen-free” in that it has essentially the same composition and structure as the ISCOM™ vaccine but without the incorporated antigen[12;14], is combined or mixed with influenza immunogen(s) such as the standard tri-valent seasonal influenza vaccine as described below. Thus, in contrast to ISCOM™ vaccines, the influenza immunogen(s) in the vaccine formulation of the present invention are not incorporated into the ISCOMATRIX™ adjuvant structure. Accordingly, the production of the vaccine formulations of the present invention is simple, robust and reproducible, and can be performed at a large scale.
Furthermore, the work leading to the present invention has demonstrated the ability of the vaccine formulation comprising standard endemic influenza immunogen(s) to protect against lethal challenge with a highly pathogenic, pandemic (H5N1) subtype of influenza A virus, using ferrets as an animal model.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.